The blood loss associated with major forms of surgery has in the past been compensated by replacement therapy, which may involve fresh frozen plasma, fresh whole blood and platelet concentrates. With recent awareness of a variety of blood borne viral infections (Hepatitis B and C, and human immunodeficiency virus, HIV), the need to reduce blood loss during surgery is a major priority. Further anxiety has been generated within National Blood Transfusion Services concerning infectivity with agents related to Bovine Spongiform Encephalitis (BSE) and Creuzfeldt-Jacob's Disease (CJD) for which there is no reliable assay at the present time.
It has been established (Royston, 1990, Blood Coagul. Fibrinol. 1:53–69; Orchard et al, 1993, Br. J. Haemat. 85:596–599) that unfettered fibrinolytic activity via the plasminogen-plasmin pathway contributes to haemorrhage and that a plasmin inhibitor such as aprotinin helps alleviate blood loss. This seems to suggest that plasmin-mediated digestion of fibrin clots and components of the coagulation system may be of primary importance as a contribution to this haemorrhagic state (Orchard et al, 1993, supra).
The use of aprotinin during cardiopulmonary bypass (CPB) surgery is now commonplace (Royston, 1990, supra; Orchard et al, 1993, supra). In particular, Orchard et al (1993, supra) have demonstrated that the bovine source inhibitor aprotinin, as the active substance in the medicament Trasylol™, reduces blood loss in CPB patients by neutralisation of plasmin activity and does not affect platelet activity. This latter finding has been confirmed by other investigators (Ray and March, 1997, Thromb. Haemost. 78:1021–1026).
Aprotinin is a well-investigated serine protease inhibitor, or ‘serpin’. It comprises 58 amino acids and acts to inhibit trypsin, α-chymotrypsin, plasmin as well as tissue and plasma kallikrein (Fritz and Wunderer, 1983, Drug Res. 33:479–494; Gebhard et al, 1986 In “Proteinase Inhibitors”, Barrett and Salvesen (eds.), Elsevier Science Publications BV pp 374–387). Aprotinin has also been found to react with thrombin and the plasminogen activators (tPA and uPA) (Willmott et al, 1995, Fibrinolysis 9:1–8).
Recent studies have shown that semi-synthetically generated homologues of aprotinin that contain other amino acids in place of lysine at position 15 of the amino acid sequence have a profile of action and specificity of action which differ distinctively from those of aprotinin (U.S. Pat. No. 4,595,674; Wenzel et al, 1985, In “Chemistry of Peptides and Proteins” Vol. 3). Some of these semi-synthetic aprotinin homologues have, for example, a strongly inhibiting action on elastase from pancreas and leucocytes. Other aprotinin homologues with arginine at position 15, alanine at position 17, and serine at position 42, are characterised by an inhibitory action which is distinctly greater than that of aprotinin on plasma kallikrein (cf. WO 89/10374).
Reference also may be made to U.S. Pat. No. 5,576,294 (Norris et al) which discloses human protease inhibitors of the same type as aprotinin. In particular, there is disclosed variants of human Kunitz-type protease inhibitor that preferentially inhibit neutrophil elastase, cathepsin G and/or proteinase 3. Compared to aprotinin, these variants have a net negative charge and are considered to have a reduced risk of kidney damage when administered to patients in large doses. In contrast, aprotinin has a nephrotoxic effect when administered in relatively high doses (Bayer, Trasylol, Inhibitor of proteinase; Glaser et al, In “Verhandlungen der Deutchen Gesellschaft Für Innere Medizin, 78. Kongress”, Bergmann, München, 1972, pp 1612–1614). This nephrotoxicity is considered to be a consequence of the strongly net positive charge of aprotinin that causes it to bind to the negatively charged surfaces of kidney tubuli.
While there is no doubt that the anti-fibrinolytic clinical use of aprotinin reduces blood loss during vascular surgery, there is evidence of increased incidence of ‘rebound thrombosis’ which manifests in graft occlusion and perioperative myocardial infarction (Van der Meer et al, 1996, Thromb. Haemost. 75:1–3; Cosgrove et al, 1992, Annals Thorac. Surg. 54:1031–1038; Samama et al, 1994, Thromb. Haemost. 71:663–669). Consistent with these findings, it has been shown that aprotinin has a somewhat broad specificity and slow tight-binding kinetic action on plasmin (Willmott et al, 1995, supra). Accordingly, the increased incidence of rebound thrombosis may be a consequence of the tight binding of aprotinin to plasmin and concomitant irreversible neutralisation of the fibrinolytic system.
Until recently, there were no effective anti-fibrinolytic agents described in the prior art with reduced propensity for causation of rebound thrombosis compared to aprotinin. However, in a recent study, Willmott et al (1995, supra) isolated and characterised a plasmin inhibitor from the venom of the Australian brown snake, Pseudonaja textilis textilis with a promising kinetic profile in respect of rebound thrombosis. This isolated preparation of plasmin inhibitor, termed Textilinin (Txln), was found to consist of a single approximately 7 kDa protein, as assessed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. In contrast to the many serine protease enzymes inhibited by aprotinin, Txln was only shown to inhibit plasmin and trypsin. It was also shown to conform to a single stage competitive reversible mechanism for the binding of plasmin. In contrast, aprotinin conforms to a two stage reversible mechanism wherein enzyme and virgin inhibitor react to initially produce a loose non-covalent complex followed by a tightly bound, stable complex in which enzyme and inhibitor remain largely unchanged (Laskowski and Kato, 1980, Annu. Rev. Biochem. 49:593–626; Travis and Salvesen, 1983, Annu. Rev. Biochem. 52:655–709; Longstaff and Gaffney, 1991, Biochemistry 30:979–986). Moreover, Txln was shown to bind plasmin more rapidly (dissociation rate constant, k1=3.85×10−5 sec−1 M−1) and with a less avid Ki (dissociation constant, Ki=1.4×10−1 M) than aprotinin (dissociation rate constant, k2=1.64×10−5 sec−1 M−1; dissociation constant, Ki=5.3×10−11 M—this latter value being in close agreement with a previously reported value of Ki=2×10−10 M (Longstaff and Gaffney, 1992, Fibrinolysis 3:89–87)). It was suggested therefore that the Txln kinetic profile may be clinically more attractive with respect to rebound thrombosis than that of aprotinin in the management of perioperative and postoperative bleeding.